Characterizing the Craniofacial Morphological Changes in Neural Crest Cell Specific Conditional Connexin‐43 Knock‐Out Mice

The craniofacial skeleton is an intricate structure that forms through complex cell‐cell interactions across neural crest and mesoderm derived cell populations. Proper cell‐cell communication during the migration and differentiation of cranial neural crest cells is critical for normal craniofacial development and this intercellular communication is thought to be facilitated through gap junctions – membrane channels composed of connexin proteins. In particular, connexin‐43 (Cx43) has been shown to be expressed in pre‐migratory and migratory cranial neural crest cells and plays a role in neural crest epithelial‐to‐mesenchyme transition. Additionally, within the skull, neural crest‐derived regions show the greatest morphological changes in mice wherein Cx43 has been globally ablated providing further evidence that Cx43 is important for proper neural crest function and ultimately proper development of the skull. To date, there have been no studies that have directly looked at the role of Cx43 in cranial neural crest cells in a mouse model. Thus, we have developed a neural crest‐specific Cx43 knockout mouse to address this gap in knowledge. In this study we (1) verify that Cx43 ablation is limited to neural crest cell and (2) characterize and compare craniofacial size and shape among wildtype, heterozygous and homozygous knockout mice. Heterozygous Wnt1‐Cre2 recombinase mice were bred with homozygous floxed Cx43 mice to create offspring which were bred to express the genotype, Wnt1‐Cre2 /+ :Cx43 fl/+ . These mice were bred to create: control mice (Wnt‐1Cre2 /+ :Cx43 +/+ ), heterozygous conditional knock‐outs (Wnt1‐Cre2 /+ :Cx43 fl/fl ) and homozygous conditional knock‐outs (Wnt1‐Cre2 +/+ :Cx43 fl/fl ). To verify the knockout, paraffin embedded sagittal sections of embryonic and post‐natal mouse hearts and skulls were tagged with Wnt1 and Cx43 antibodies. RT‐PCR, and Western blotting were performed on neural crest and mesoderm derived newborn skull bones, to verify the expression levels of Cx43 mRNA and protein respectively. Newborn skulls from each genotype were imaged using high resolution micro‐CT and these images were used to quantify and compare skull morphology among genotypes using statistical analyses of shape. We have confirmed that Cx43 ablation is limited to neural crest cells in our mice. Analyses of skull shape comparisons among genotypes is ongoing with our initial findings indicating that mice homozygous for the knockout demonstrate overt changes to skull morphology, heterozygous mouse skulls are qualitatively similar to control mouse skulls. We predict that statistical analyses will uncover that only the bones derived from neural crest cells will display morphological differences among genotypes. Our study addresses a fundamental gap of the role connexin‐43 plays in cranial neural crest function and subsequent development of much of the skull. Support or Funding Information Funding: Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (R5211A02)

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